Enhanced Release of Molecules upon Ultraviolet (UV) Light Irradiation

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Enhanced Release of Molecules upon UV Light Irradiation from Photoresponsive Hydrogels Prepared from Bifunctional Azobenzene and Four-Arm Poly(ethylene glycol) Shiva Kumar Rastogi, Hailee E. Anderson, Joseph Lamas, Scott L. Barrett, Travis Cantu, Stefan Zauscher, William J. Brittain, and Tania Betancourt ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16183 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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ACS Applied Materials & Interfaces

Enhanced Release of Molecules upon UV Light Irradiation from Photoresponsive Hydrogels Prepared from Bifunctional Azobenzene and Four-Arm Poly(ethylene glycol) Shiva K. Rastogiξ,*, Hailee E. Andersonξ, Joseph Lamas‡, Scott Barretξ, Travis Cantu†, Stefan Zauscher‡, William J. Brittainξ and Tania Betancourtξ ,† ξ

Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, United States of America

†

Materials Science, Engineering, and Commercialization Program, Texas State University, San Marcos, TX, United States of America

‡

Department of Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina, United States of America

KEYWORDS: Four-Arm-PEG-NH2, 4,4’-di-NHS azobenzoic ester, photoresponsive hydrogel, azobenzene photoisomerization, Alexa Fluor dye, drug delivery, and triggered release. ABSTRACT: Advances in biosensors and drug delivery depend on hydrogels that respond to external stimuli. In this work we describe the preparation and characterization of photoresponsive hydrogels prepared by crosslinking of di-NHS ester of azobenzoic acid and four-armed, amine-terminated poly(ethylene glycol). The porous structure and composition of the hydrogels were confirmed by scanning electron microscopy and Fourier transform infrared (FTIR) spectroscopy. The reversible photoisomerization of the azobenzene-containing hydrogel crosslinkers in the gels was confirmed by absorption spectroscopy. Specifically, the photoisomerization of the crosslinkers between their trans and cis configurations was observed by monitoring the absorbance of the hydrogels at the two characteristic peaks of azobenzene (π-π* at 330 nm and n-π* at 435 nm). The effect of photoisomerization on the hydrogel structure was investigated by microscopy. Ultraviolet (UV) irradiation-induced reduction in hydrogel size was observed which may be a result of the inherently smaller footprint of the cis azobenzene conformation as well as dipole-dipole interactions between the polar cis azobenzene and the polymer network. The UV-triggered reduction in hydrogel size was accompanied by enhanced release of the near infrared fluorescent dye Alexa Fluor® 750 (AF750). Enhanced release of AF750 was observed in samples irradiated with UV versus dark control. Together these data demonstrate the potential of these systems as reversible photoresponsive biomaterials.

INTRODUCTION Hydrogels are crosslinked networks of hydrophilic or amphiphilic polymers that are able to imbibe a large amount of water.1 Due to their large water content, these materials have found applications in a wide range of biomedical systems including tissue engineering scaffolds, contact lenses, wound healing materials, biosensors, and drug delivery systems.2-6 The development of intelligent hydrogels that can respond to internal or external stimuli has been the focus of research over the past few decades.78 Hydrogels can be tailored to respond to different stimuli with proper selection of polymer composition and assembly. Significant progress has occurred in the development of hydrogels that respond to changes in the pH and temperature of the surrounding tissues.9 pHresponsive hydrogels typically include ionizable functional groups that can change between charged and uncharged configuration depending on acidity level of the surrounding tissue. These pH-responsive hydrogels are

able to swell when charged due to charge repulsion between adjacent polymer strands.10 While these systems are very promising, hydrogels that can respond to exogenous stimuli such as light, magnetic fields, electric fields, or ultrasound waves can open the doors to applications in which user control can enable noninvasive or minimally-invasive function with spatial and temporal control.8, 11-13 Photoresponsive biomaterials have found applications as drug delivery systems, biosensor components, and even in biodegradable scaffolds. The many mechanisms utilized for light-triggered biomaterial response has been previously reviewed,8, 14-15 but most photoresponsive biomaterials incorporate photocleavable or photoisomerizable components that, when incorporated in properly designed systems, lead to light-mediated changes in the properties of the biomaterial. Depending on the design of the system, light-induced responses typically lead to material degradation or permanent

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chemical modification. For example, photodegradable hydrogels have been prepared by free radical polymerization utilizing a photocleavable nitrobenzyl ether-modified difunctional crosslinker.16 Similarly, hydrogels with photocontrollable presentation of cell adhesion molecules were also prepared utilizing the same photocleavable moiety.16 Azobenzene has been by far the most widely used photosensitive component of photoresponsive biomaterials due to a nearly degradation-free and reversible isomerization between cis and trans isomers. Photoisomerization can be accomplished by selective excitation of trans (300-380 nm) or cis (>400 nm) conformation. In addition, the metastable cis isomer also undergoes thermal isomerization to trans. Our group has previously demonstrated the formation of photoresponsive micelles prepared from amphiphilic DNA-azobenzene conjugates.17 Photoresponsive biomaterials utilizing azobenzene-grafted nucleic acids have also been previously demonstrated. In these materials, photoisomerization of azobenzene disrupts hybridization of complementary strands, leading to disruption of noncovalent crosslinks. Among these materials, photocontrollable DNA nanostructures in which UVinduced photoisomerization of azobenzene-grafted DNA leads to changes in the three-dimensional geometry of the self-assembled macromolecules have been reported.18 Similarly, photoresponsive DNA-crosslinked hydrogels in which photoisomerization of azobenzene-grafted DNA induces hydrogel disassembly were shown to act as lightcontrolled systems for the delivery of small molecules, proteins, and nanoparticles.13 Azobenzene-grafted DNA has also been used as a molecular gate for the release of encapsulated agents from mesoporours silica nanoparticles upon UV light irradiation.19 Azobenzene has also been used to enable light-mediated disruption of guest-host interactions utilized for the preparation of self-assembled biomaterials.20 In these systems, UV light-induced photoisomerization of azobenzene results in dissociation of azobenzenecyclodextrin complexes, leading to biomaterial disassembly. The preparation of photoresponsive hydrogels from cyclodextrin modified polymeric glucose (curdlan) and azobenzene-grafted poly(acrylic acid) was demonstrated by Tamesue et al.21 Similarly, photoresponsive hydrogels made from azobenzenemodified dextran and cyclodextrin-modified dextran were shown to enable UV light-controlled release of encapsulated protein.22 Although numerous natural and synthetic polymers have been utilized for the preparation of hydrogels for biomedical applications, poly(ethylene glycol) (PEG) has been one of the most widely used due to its high biocompatibility, lack of immunogenicity, and resistance to protein adsorption.23 In addition, PEGs are commercially available in both linear and multi-arm forms and can be readily functionalized at their termini for use as precursors for hydrogel preparation. In this work we describe the preparation and characterization of photo-responsive hydrogels (PR-Hgels) made from branched PEG crosslinked by a bifunctional

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photoresponsive azobenzene linker (Scheme 1), and demonstrate their application as systems for lighttriggered release of a model encapsulated agent. Compared to the various systems described above, the PR-Hgels herein described undergo light-induced changes that do not lead to degradation or disruption of crosslinks. Instead, exposure of the PR-Hgels to UV and visible light results in crosslinker photoisomerization that can be used to reversibly control hydrogel swelling and contraction, respectively, and therefore enable applications in drug delivery and biosensing, among others. EXPERIMENTAL SECTION Materials. Unless otherwise noted, all reagents and solvents were purchased from commercial sources (Acros Organics and Sigma-Aldrich, USA) and used without purification. 4-Arm-PEG-Amine (Mol. Wt. 20,000) was obtained from Laysan Bio. Inc. (Arab, AL). Alexa Fluor® 750 NHS ester (AF750) was obtained from Thermo Fisher Scientific (Waltham, MA). Dry solvents were obtained using a solvent purification system (Innovative Technology, Inc.). Synthesis of Hydrogel Precursors. Chemical reactions were performed in oven-dried flasks either open to air or under nitrogen gas. Reaction progress was monitored by thin layer chromatography (TLC). TLC was performed on pre-coated silica gel 60 F254 (250 μm) with UV light (254 nm). Flash column chromatography was performed on silica gel (32-63 μm, 60 Å pore size). Synthesis of Azobenzoic acid (2). (E)-4,4’-(diazene-1,2diyl)dibenzoic acid (2) was prepared as previously reported.24-25 In brief, p-nitrobenzoic acid (1.5 g, 6.75 mmol) and NaOH (5.1 g, 0.125 mmol) were mixed in water (25 mL). This solution was heated on a water bath until the solid dissolved. A hot aqueous glucose solution (10.1 g in 20 mL of water) was then added slowly into the above mixture at 50 °C whereupon a yellow precipitate was obtained. This solution immediately changed to a brown solution upon further addition of glucose. Then, a stream of air was passed into the mixture for 3 h and a light brown precipitate was obtained. This precipitate was filtered, dissolved in water, and acidified with acetic acid (5 mL) at which point a light pink precipitate was obtained. This precipitate was filtered, washed with water (50 mL), and dried in a desiccator to obtain 2 (yield 0.75 g, 67%) as a brownish orange powder. 1H NMR (DMSOd6): δ 8.17 (d, J = 8.5 Hz, 4H, Ar-H) and 8.02 (d, J = 8.5 Hz, 4H, Ar-H). Synthesis of di-NHS Ester (3). (E)-bis(2,5-dioxo-1pyrrolidin-1-yl)4,4’-(diazene-1,2-diyl)dibenzoate (3) was prepared by a previously reported method,24 from 2. Compound 2 (0.108 g, 0.4 mmol) was dissolved in dimethylformamide (DMF) at 60 °C. A catalytic amount of 4-dimethylaminopyridine (DMAP) (0.015 g; 0.123 mmol), N-hydroxysuccinimide (NHS) (0.117 g, 1.02 mmol), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl) (0.151 g, 0.786 mmol) were then added sequentially to the solution. The reaction was stirred at room temperature for 24 h. DI water (20 mL)


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was added to the solution until a red precipitate was obtained and filtered. The solid was washed with water (50 mL) several times and dried to yield a light red product 3 (yield 0.145 g, 78%). IR (KBr): 3072 cm-1 (νC-H, aromatic), 1692 cm-1 (νC=O), 1603 cm-1 (νC=C), 1580 cm-1 (νN=N), 1299 cm-1 (νC-N). 1H NMR (DMSO-d6): δ 8.36 (d, J = 8.4 Hz, 4H, Ar-H) and 8.19 (d, J = 8.4 Hz, 4H, Ar-H), 2.92 (s, 8H, CH2). λmax (E) = 330, 470 nm, λmax (Z) = 330, 445 nm.

NMR spectra were recorded on Bruker Avance III 400 spectrometer. 1H and 13C NMR shifts are reported relative to tetramethylsilane (TMS, 400.13 MHz for 1H; 100.61 MHz for 13C). Chemical shifts (δ) are reported in ppm relative to the TMS internal standard. Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and coupling constant (J) in Hz. FTIR spectra were recorded on KBr pellets using a Tensor II, Bruker instrument and spectra were analyzed using OPUS ver. 6.5 software.

Scheme 1. Synthesis of photo-responsive hydrogels (PR-Hgels) Preparation of Photoresponsive Hydrogels (PRHgels). Hydrogel preparation was carried out as shown in Scheme 1. Specifically, 4-Arm-PEG-Amine (4) (13.5 mg) was dissolved in DI water (50 μL) in a plastic microcentrifuge tube to make a 13.5 mM solution. Separately, di-NHS ester of azobenzoic acid (3) (1.6 mg) was dissolved in dry DMF (100 μL) in another plastic microcentrifuge tube to make a 34.5 mM solution. The two solutions were mixed together at a 1:1 volume ratio and vortexed for 2 min. The resulting mixture was used to prepare five different sizes of PR-Hgels by pipetting 0.3, 0.5, 1, 2 or 50 μL of the mixture onto silanized (Sigmacote®- treated) or bare glass cover-slips. The droplets were left in a humidified container for 1 h at room temperature and then cured either at room temperature or at 37 °C for 16 h to yield orange colored dry PR-Hgels. IR (KBr): 3446 cm-1 (νN-H), 2918 cm-1 (νC-H Aromatic), 1739 cm-1 (νC=O), 1652 cm-1 (νC=C), 1576 cm-1 (νN=N), 1305 cm-1 (νC-N), 1254 cm-1 (νC-O). λmax (E) = 330 nm, λmax (Z) = 445 nm. PR-Hgel Photoisomerization and Light-Induced Swelling. Dry PR-Hgels (1 mm in diameter) were placed on the center of the grid of a disposable hemocytometer (C-Chip-DHC-N01; Incyto, Korea) and bright field microscopy images were taken with an EVOS® FL

(Advance Microscopic Group) digital microscope. The dry gels were then soaked in water (100 μL) and kept in humid chamber for 24 h. After 24 h, the gels were washed twice with water (100 μL). The wet, swollen gels were then exposed to UV followed by visible light for 15 min each and images were taken before and after light exposure. Photoshop was utilized to quantitate the changes in area of the PR-Hgels from the images. Specifically, using a calibration of number of pixels per mm2, changes in the area of the gels upon UV or visible light exporsure were quantified by determining the number of pixels within an encircled area corresponding to the perimeter of the gels. Encapsulation of Alexa Fluor 750® (AF750) Dye in PRHgels. AF750 was dissolved in water at a concentration of 128 μM and maintained at room temperature for over 24 h to enable hydrolysis of N-hydroxysuccinimide ester (amine reactive) functional group. Dry PR-Hgels (1 mm in diameter) prepared from 1 μL of reaction mixture (1:1 v/v mixture of 13.24 mM 4 and 33.8 mM 3) were soaked in an aqueous solution of AF750 (50 μL; 128 μM) in a microcentrifuge tube for 24 h at RT. After that, the dye solution was removed and swollen gels were washed twice with water (100 μL) for 10 min each time. Release of AF750 from PR-Hgels. The washed AF750loaded PR-Hgels were immersed in 500 μL of water and

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exposed continuously to either UV, ambient, or a sequence of ambient and UV light. The release of the dye was monitored by collecting aliquots of the aqueous solution every minute. For these studies, a fiber-Lite MH100A light source (Dolan-Jenner Industries, Boxborough MA, 01719) and a UV colored glass filter (see supplemental Figure S1 for filter transmission characteristics) were used to shine UV light on the PR-Hgel. The distance from light source to the sample was kept at 6 in to avoid heating the sample. Release of fluorescent dye from hydrogels was measured by fluorescence spectroscopy using a Biotek® Synergy H4 Hybrid Multi-Mode Microplate Reader together with a Take3 microvolume plate.

azobenzoic acid and 4-Arm amine-terminated PEG. The size and shape of the PR-Hgel samples varied depending on the curing surface and temperature. We observed a larger decrease in size upon gel curing (relative to uncured liquid drop) for curing on hydrophilic surfaces (non-Sigmacote® treated glass cover slides) compared to the more hydrophobic Sigmacote®-treated glass cover slides (Figure 1). Curing of PR-Hgels at room temperature resulted in smooth PR-Hgels (Figures 1a,b) while curing at 37 °C caused drying (coffee ring) effects leading to heterogeneous gels (Figures 1c,d). Based on these results, all further studies were conducted with PR-Hgels prepared on Sigmacote®-treated glass coverslips cured at room temperature.

UV-Vis Absorption and Photoisomerization of Solutions. Absorption measurements were performed using either a Cary 100 Bio UV-Vis spectrometer or an Ocean Optics component system with a HR2000+ detector, qpodTM sample holder, DH-2000 light source and Spectra Suite software for data collection. Solutions were degassed via 3 freeze-pump-thaw cycles. Photochemical kinetic experiments used an Oriel 66901 50–200 W Research Arc Lamp; the output light was cooled with a water filter (Newport 6117) that removes infrared light. See supplemental Figure S1 for transmission characteristics of UV bandpass and vis pass filters.

The morphology of PR-Hgels was investigated using scanning electron microscopy. Figures 2a-c show the characteristic network structures of the PR-Hgels under three magnifications from 20 to 200 μm. The PR-Hgels show a high level of porosity similar to that of previously reported PEG-based hydrogels.26

UV-Vis Absorption and Photoisomerization of PRHgels. UV-Visible spectra of hydrogels or solutions extracted from the hydrogels were recorded using a Biotek® Synergy H4 Hybrid Multi-Mode Microplate Reader. Spectra and data were analyzed using Gen5 software ver 2.00.15. Scanning Electron Microscopy (SEM) of PR-Hgels. SEM samples were prepared by drop casting the hydrogels onto a Si wafer, followed by freezing in liquid nitrogen and lyophilization with a Labconco FreeZone 4.5L freeze dryer. The hydrogels were then sputter coated with a 2 nm thick iridium layer using an EMS Quorum EMS150T ES sputter coater. The samples were then imaged using a Helios NanoLab 400 FEI scanning electron microscope at a working distance of 5 mm and at 5 kV. Rheology Analysis. Rheological measurements of swollen hydrogels were performed by loading the gels onto a rheometer (TA instruments, AR G2) with an 8-mm parallel plate geometry. When exposing the hydrogels to UV, the geometry was lifted and a flashlight with a UV bulb (365 nm, 12-LED ultraviolet flashlight, American’s Preferred) was used to irradiate the gels for 15 min. Each measurement was performed at 24 oC using an oscillatory strain of 5% and a frequency of 5 rad/s (linear viscoelastic region). The gels were immersed in water before each measurement and during UV exposure to maintain swelling equilibrium. Gels larger than the geometry were cut using an 8-mm Biopunch while in their swollen state. RESULTS AND DISCUSSION PR-Hgel Preparation. PR-Hgel networks were formed by the amide bond cross-linking between di-NHS ester of

Figure 1. PR-Hgels prepared from 1 μL of reaction mixture on Sigmacote®-treated square coverslips (a and c) and on bare (non-Sigmacoate®-treated) circular glass coverslips (b and d). PR-Hgels on (a and b) were cured at room temperature and on (c and d) at 37 °C.

The functional groups present in PR-Hgels were characterized by FTIR spectroscopy using KBr pellets. The FTIR spectra in Figure 3 shows the infared absorptions for N-H at 3446 cm-1, aromatic C-H at 2918 cm-1, carbonyl (C=O) peak at 1739 cm-1, C=C at 1652 cm-1, N=N at 1576 cm1 , C-N at 1315, 1305 cm-1 and C-O at 1254 cm-1. These peaks indicate the presence of azobenzene and PEG moieties in PR-Hgels. Further, this spectrum shows absorptions corresponding to functional groups from both precursors 3 and 4 (supplemental Figure S2a-b). The actual content of azobenzene in the PR-Hgels was determined indirectly by extracting unreacted azobenzene from the gels and quantifying the amount of extracted azobenzene by absorption spectroscopy utilizing a standard curve (supplemental Figure S3).


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Based on this study, it was determined that 97% of the azobenzene added in the feed was reacted aand became part of the hydrogel network. Accordingly, the network consists of approximately 0.56% azobenzene on a molar basis or 3.2% azobenzene on a mass basis relative to PEG ethylene glycol units. The photoisomerization properties of azobenzene crosslinked PR-Hgels were characterized using UV-vis absorption spectroscopy. Two characteristic peaks (π-π* at 330 nm and n-π* at 445 nm) of azobenzene were observed in the absorption spectra of wet PR-Hgels (blue line; Figure 4a). Similar UV-vis spectra were observed for

the PR-Hgels and DMSO solution of azobenzene 3 after UV and vis irradiation (Figure 4). Based on presumption that gel extraction removed all unreacted azobenzene 3, the UV-vis spectra represent azobenzene groups covalently bound in the network. The 330 nm absorption band (π-π*) decreased by 17% for a PR-Hgel sample exposed to UV and a slightly larger decrese of 26% was observed for the solution of 3. This difference in cis/trans ratio at the photostationary state is consistent with a more constrained molecular environment in the PR-Hgel that hinders azobenzene isomerization.

Figure 2. SEM images of PR-Hgels at different magnifications. Scale bars in the images represent (a) 200 μm, (b) 50 μm (sample tilted 30°), and (c) 20 μm. PR-Hgel in (b) was tilted 30°.

Figure 3. FTIR spectrum of PR-Hgels. Blue and black peak labels denote azobenzene and PEG vibrational assignments.



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Figure 4. UV-visible absorption spectra of (a) PR-Hgels and (b) di-NHS ester of azobenzoic acid (3) in solution (1.6 x 10-3 M in DMSO) before irradiation (blue curve), after irradiation with UV (red curve), and after vis irradiation (green curve).

Figure 5. Reversibility of PR-Hgel azobenzene photoisomerization.

The photoisomerization of azobenzene between cis- and trans-isomers in PR-Hgels was confirmed by irradiating the wet PR-Hgels with UV and visible light 15 min each time. For this experiment PR-Hgels (prepared from 3 μL of reaction mixture) were soaked in water (200 μL) in a 96-well plate overnight and washed three times with water prior to light exposure. Then, the PR-Hgels were exposed to alternating UV and visible light for 15 min each. After every 15 min of light exposure, the absorbance of the PR-Hgels at 330 nm was recorded. The consistent change in absorbance with repeated photocycles depicted in Figure 5 demonstrates that photoisomerization of the PR-Hgel is reversible with no significant degradation. We studied the kinetics of the thermal isomerization from trans to cis for both DMSO solutions of 3 and the PR-Hgel (supplemental Figures S4 and S5). We observed extremely fast isomerization for solutions of 3 consistent with a literature report of a 23 s half-life for the cis isomer of structurally related azobenezenes (4,4’dicarbonyl azobenzene).27 This rapid isomerization made it difficult to obtain detailed a calibration for cis/trans ratio (as determined by 1H NMR, see Figure S4) to the UV-Vis spectra of mixtures. The rate of thermal isomerization for the PR-Hgel (supplemental Figure S5) was measured by UV-Vis; the rate

corresponds to a 1 h half-life for the azobenzene crosslinked unit in the gel. There are two major findings from these results. First, covalent incorporation of a 4,4’-dicarbonyl substituted azobenzene into the network structure decreases the thermal isomerization rate of the cis structural unit by two orders of magnitude compared to solution. Second, only a fraction (20% increase in cis population for two 15-min exposures) of the azobenzene units undergo isomerization to cis and this is sufficient to cause enhanced release. The effect of UV and visible light on the diameter of PRHgel disc-shaped samples was analyzed by microscopy. Figure 6 shows triplicate results of these experiments, where the dry PR-Hgels ~1 mm in size (prepared from 1 μL of reaction mixture) were placed in the center of disposable plastic haemocytomer grids (Figure 6a) and soaked in water (50 μL) overnight, washed three times with water, and imaged (Figure 6b). These wet gels were approximately double in size (~2 mm) relative to their dry size. The wet gels were irradiated with UV for 15 min and imaged immediately; upon UV irradiation, the gels were found to decrease in size to an average of 77% of their initial area (Figure 6c) as determined via image analysis. Upon visible light irradiation, the PRHgels regained their size (Figure 6d), showing that


6

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~98% of the gel sample area could be recovered. It should be noted that the dark spheres on Gel #1 and #2 in columns c and d of this figure are artifacts from air bubbles that were in the solution surrounding the hydrogels. These experiments clearly indicate the PRHgels change in size upon exposure to UV and visible light. We assert that this macroscopic change in PRHgel diameter is coupled to the molecular photoisomerization of the azobenzene crosslinkers. UVinduced cis formation decreases the sample diameter which is recovered by visible irradiation. We speculate

that this response is associated with the inherently smaller footprint of the azobenzene crosslinkers in the cis conformation as well as with the introduction of dipole-dipole interactions between the polar cis azobenzene and the polymer network. For the parent azobenzene compound, the dipole moment increases 4.6 D and the end-to-end distance between paracarbons decreases by 0.35 nm upon isomerization to cis28 which would lead to a sizable decrease in the length of the azobenzene crosslinker.

Figure 6. Brightfield microscopy images of PR-Hgels on a haemocytometer grid showing light-induced response (decreasing diameter with UV vs. increasing diameter with visible irradiation). White scale bars represent a distance of 2 mm. Gels 1a–3a are dry PR-Hgels, Gels 1b-3b are hydrated gels before UV, Gel 1c-3c are hydrated gels after UV, and Gels 1d-3d are hydrated gels after visible light. Note: dark spheres on Gel # 1 and # 2 in columns c and d are artifacts from air bubbles.

To elucidate the viscoelastic properties of the PR-Hgels and their response to UV light, the shear modulus was first measured between 1 rad/sec and 100 rad/sec with a constant strain of 5% (Figure 7, Top). In this frequency range, the storage modulus, G’, of the gels was significantly higher than of the loss modulus, G”, regardless of irradiation status. These results suggest that the elastic properties of the gels dominate and are relatively constant throughout this frequency range. In addition, as shown in Figure 7a, we were unable to detect

significant changes in either G’ or G” upon UV irradiation, suggesting UV irradiation has little effect on the mechanical properties of the gels. Figure 7b shows how the gels behave as the strain is increased from 1% to 100%. The behavior shown indicates that the mechanical properties of the gels are independent of strain until the strain reaches ~63%. As with the frequency sweep, there was no measurable difference between the viscoelastic properties of the gels when exposed to UV light in the


ACS Applied Materials & Interfaces strain sweep. The relatively constant value of the storage modulus suggests that the gels were heavily crosslinked. These results contrast the photoresponsive changes in gel modulus reported by Anseth and co-workers.29 UV irradiation caused a 100-200 Pa decrease in storage modulus. We speculate that the network structure of PR-Hgel is significantly different that Anseth’s hydrogel that was prepared by the Michael reaction of a peptide crossslinker containing azobenzene and 4- or 6-arm PEG vinyl sulfone. Further investigation is warranted to fully understand how UV affects the mechanical properties of these PR-Hgels. Nonetheless, our microscopy data does show significant changes in the size of the PR-Hgels, which are expected to cause modest changes in the mechanical properties of the gels.

the size of CD-containing hydrogels decreased while a PR-Hgel without CD increased in size. Inspection of the paper’s figures indicates dimensional changes of 10-15% in the diagonal distance of a hydrogel cuboid surface.30 The size increase for non-CD PR-Hgels is opposite to our results. Harada, et al. hypothesized that trans azobenzene units are aggregated in the hydrogels and upon UV exposure, conversion to cis has the net effect of decreasing effective crosslink density and, thereby, increasing the gel size.30 We argue that trans aggregation is not occuring in our PR-Hgel because we do not observe significant shifts in UV-Vis peak maxima where ~10 nm shifts are expected for reversibly aggregated systems. While there is some structural similarity to the PR hydrogels reported in the literature,21,28,29 we are among the first to examine potential applications of these sytems in drug delivery. The encapsulation of AF750 was used to demonstrate that the loading of small organic molecules into PR-Hgels was possible. The release of the dye with or without irradiation with UV was studied. First, preliminary studies were carried out to ensure that the AF750 dye would not be photobleached upon exposure to UV or visible light in the conditions of the release studies. As shown in supplemental Figure S6, no photobleaching is observed. Next, a calibration curve of AF750 fluorescence with concentration was constructed (supplemental Figure S7) to enable quantification of the released dye. For dye loading and release experiments three dry PR-Hgels of 1mm diameter were soaked in an aqueous solution (128 μM) of AF750 for 24 hours at room temperature. After that, the samples were washed with water three times to remove un-entrapped dye. Next, the hydrogels were immersed in 500 μL of water and exposed continuously to either UV or ambient light. The release of the dye was monitored by collecting aliquots of the aqueous solution every minute and analyzing them via fluorescence spectroscopy. The samples that were irradiated with UV showed approximately two-fold higher release of AF750 than the control PR-Hgels (Figure 8). 0.12

Figure 7. Storage modulus G’ and loss modulus G’’ (in Pa) of swollen PR-hydrogels in the absence of irradiation and upon UV irradiation. (A) Frequency sweep from 1 – 100 rad/s. (B) Strain sweep between 0 and 100%.

Harada and co-workers have reported photoresponsive hydrogels containing azobenzene where gel assembly is mediated by molecular recognition between azobenzene pendant groups and tethered cyclodextrins.21, 30 This gel self-assembly was used to create reversible gelation21 and hydrogels that show fast response dimensional changes.30 In the 2016 report, photoresponsive gels were synthesized using azobenzene grafted to cyclodextrin (CD) and an NHS-terminated PEG crosslinker.30 Upon UV exposure,

Cumulative AF750 Released (nmol)

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9

10

Figure 8. Release of AF750 dye from UV irradiated (“cis” rich) sample and ambient light exposed control (“trans”

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rich). Data points correspond to the average of independent experiments. Error bars correspond to the standard deviation between samples.

Finally, separate studies were conducted in which samples were first irradiated with ambient light for 3 minutes, followed by UV light exposure (Figure 9). UV light results in an immediate increase in the mass of dye released. We argue that the enhanced release of AF750 dye in the presence of UV is coupled to the trans-to-cis photoisomerization and the decrease in sample diameter. The change in polarity and conformational change with formation of cis azobenzene crosslinkers increases the partitioning of the dye into aqueous phase. 0.14 Cumulative AF750 Released (nmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.12 0.1 0.08 0.06 0.04

cyclodextrin-grafted poly(acrylic acid).34 Further studies would need to be conducted to determine if such azobenzene derivatives would also cause changes in the size of the hydrogels. CONCLUSION In this work we describe the preparation and characterization of azobenzene-crosslinked photoresponsive hydrogels from multi-armed PEG precursors. Porous hydrogels were formed by the reaction of the di-NHS ester of azobenzoic acid and amineterminated four-arm PEG. A UV-induced decrease in hydrogel diameter was observed. We believe this macroscopic diameter decrease is coupled to azobenzene photoisomerization. For samples imbibed with dye, this phenomenon effected a significant increase in AF750 release upon UV exposure. Future work will focus on the evaluation of the effect of polymer precursor size and structure on the photoresponsiveness of the hydrogels, as well as on development of alternative hydrogel systems that can respond to red or near-infrared light.

Ambient

0.02

ASSOCIATED CONTENT

Ambient + UV

Supporting Information

0 0

1

2

3

4 5 6 Time (min)

7

8

9

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The Supporting Information is available free of charge on the ACS Publications website. (PDF) Figure S1 – S7 (pdf)

Figure 9. Cumulative release of AF750 from PR-Hgels after either continuous irradiation with ambient light, or ambient light for 3 minutes followed by UV light for 7 minutes. Gels were suspended in 500 µL of DI water and 50 µL of aliquots were removed every minute for AF750 quantification by fluorescence spectroscopy. Data points correspond to the average of independent experiments. Error bars correspond to the standard deviation between samples.

The work presented shows the potential of azobenzenecrosslinked hydrogels as UV-triggered systems for drug delivery or biosensors. For in vivo use, these systems or their application will need to be modified to accommodate the use of red or near-infrared light as a trigger. Longer wavelengths are able to penetrate more deeply into tissue as a result of the low absorption, scattering and fluorescence of biological chromophores and tissues in this range.31-32 To achieve this, the hydrogels could be embedded with up-converting nanoparticles as previously done for photocleavable, o-nitrobenzyl crosslinked gels.33 Alternatively, an azobenzene derivative that photoisomerizes upon exposure to longer wavelengths could be utilized. For example, Want et al. demonstrated the preparation of red-light responsive hydrogels through the functionalization of poly(acrylic acid) with tetra-ortho-methoxy-substituted azobenzene and the supramolecular assembly of this system with

AUTHOR INFORMATION Corresponding Author *Shiva K. Rastogi, Department of Chemistry and Biochemistry, Texas State University, [email protected].

Author Contributions All authors made contributions to manuscript preparation and given final approval for publication.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Special thanks go to Mr. Mark Riggs for his help with microscopy imaging. We also thank the National Science Foundation (PREM Center for Interfaces, DMR-1205670) for financial support of this research. The authors also acknowledge the support of the National Science Foundation for NMR instrumentation (CR1IF:MU-0946998 and MRI-0821254). We also acknowledge support from the Robert A. Welch Foundation (AI-0045), and the Texas Emerging Technology Fund.

ABBREVIATIONS AF750 DI DMF DMAP

Alexa Fluor® 750 deionized dimethylformamide 4-dimethylaminopyridine

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ACS Applied Materials & Interfaces EDC.HCl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FTIR G’ G’’ NHS Pa PEG PR-Hgel NMR SEM TLC vis UV

N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride Fourier transform infrared spectroscopy storage modulus (Pa) loss modulus (Pa) N-hydroxysuccinimide Pascals poly(ethylene glycol) photoresponsive hydrogel nuclear magnetic resonance scanning electron microscopy thin layer chromatography visible ultraviolet

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Scheme 1. Synthesis of photo-responsive hydrogels (PR-Hgels) 252x123mm (300 x 300 DPI)


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Figure 1. PR-Hgels prepared from 1 µL of reaction mixture on Sigmacote®-treated square coverslips (a and c) and on bare (non-Sigmacoate®-treated) circular glass coverslips (b and d). PR-Hgels on (a and b) were cured at room temperature and on (c and d) at 37 °C. 87x91mm (300 x 300 DPI)



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Figure 2. SEM images of PR-Hgels at different magnifications. Scale bars in the images represent (a) 200 µm, (b) 50 µm (sample tilted 30°), and (c) 20 µm. PR-Hgel in (b) was tilted 30°. 58x18mm (300 x 300 DPI)


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Figure 3. FTIR spectrum of PR-Hgels. Blue and black peak labels denote azobenzene and PEG vibrational assignments. 109x62mm (300 x 300 DPI)



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Figure 4. UV-visible absorption spectra of (a) PR-Hgels and (b) di-NHS ester of azobenzoic acid (3) in solution (1.6 x 10-3 M in DMSO) before irradiation (blue curve), after irradiation with UV (red curve), and after vis irradiation (green curve). 143x59mm (96 x 96 DPI)


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Figure 5. Reversibility of PR-Hgel azobenzene photoisomerization. 64x35mm (300 x 300 DPI)



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Figure 6. Brightfield microscopy images of PR-Hgels on a haemocytometer grid showing light-induced response (decreasing diameter with UV vs. increasing diameter with visible irradiation). White scale bars represent a distance of 2 mm. Gels 1a–3a are dry PR-Hgels, Gels 1b-3b are hydrated gels before UV, Gel 1c-3c are hydrated gels after UV, and Gels 1d-3d are hydrated gels after visible light. Note: dark spheres on Gel # 1 and # 2 in columns c and d are artifacts from air bubbles. 152x111mm (300 x 300 DPI)


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Figure 7. Storage modulus G’ and loss modulus G’’ (in Pa) of swollen PR-hydrogels in the absence of irradiation and upon UV irradiation. (A) Frequency sweep from 1 – 100 rad/s. (B) Strain sweep between 0 and 100%. 119x171mm (300 x 300 DPI)



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Figure 8. Release of AF750 dye from UV irradiated (“cis” rich) sample and ambient light exposed control (“trans” rich). Data points correspond to the average of independent experiments. Error bars correspond to the standard deviation between samples. 64x49mm (300 x 300 DPI)


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Figure 9. Cumulative release of AF750 from PR-Hgels after either continuous irradiation with ambient light, or ambient light for 3 minutes followed by UV light for 7 minutes. Gels were suspended in 500 µL of DI water and 50 µL of aliquots were removed every minute for AF750 quantification by fluo-rescence spectroscopy. Data points correspond to the aver-age of independent experiments. Error bars correspond to the standard deviation between samples. 58x44mm (300 x 300 DPI)



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Graphical Abstract 109x47mm (300 x 300 DPI)


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Enhanced Release of Molecules upon Ultraviolet (UV) Light Irradiation

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